Association of EGLN2 rs10680577 Polymorphism with the Risk and Clinicopathological Features of Patients with Prostate Cancer

Several studies have evaluated the association between EGLN2 4-bp insertion/deletion (ins/del) polymorphism (rs10680577) and many cancers. However, up to date, no study has inspected the impact of rs10680577 polymorphism on prostate cancer (PCa) risk. This case-control study was achieved on 170 pathologically confirmed PCa patients and 196 cancer free men to inspect whether rs10680577 variant is related to the risk and clinicopathological features of patients with PCa. Genotyping was performed by mismatched polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The findings did not support an association between the variant with the risk and clinicopathological characteristics of PCa patients. When we pooled our results with six preceding studies, the findings suggested that rs10680577 variant significantly augmented the risk of overall cancer in heterozygous (OR=1.38, 95 % CI=1.26-1.52, p<0.00001, ins/del vs ins/ins), homozygous (OR=1.66, 95 % CI=1.05-2.61, p=0.029, del/del vs ins/ins), codominant (OR=1.44, 95%CI=1.32-1.58, p<0.00001, ins/del+del/del vs ins/ins), and allele (OR=1.32, 95%CI=1.18-1.49, p<0.00001, del vs ins) genetic models. Additional well designed studies with larger sample sizes are necessary to confirm our findings.


Introduction
Prostate cancer (PCa) is one of the most common cancer among men globally (Jemal et al., 2011). The precise mechanisms underlying PCa development is largely unknown. Mounting evidence suggests that genomic and environmental factors play a role in development and progression of PCa (Cunningham et al., 2003;Chokkalingam et al., 2007;Zhou et al., 2015;Sattarifard et al., 2018). Small insertions/deletions (indels), the second most common form of genetic variations in human genome, have been linked to cancer development (Mullaney et al., 2010;Hashemi et al., 2018a;Hashemi et al., 2018c;Hashemi et al., 2018d).
Hypoxia, a main characteristic of solid tumors, leads to alterations of gene expression in tumor cells to adapt to the hypoxic environment (Brahimi-Horn et al., 2007). The

Abstract
Several studies have evaluated the association between EGLN2 4-bp insertion/deletion (ins/del) polymorphism (rs10680577) and many cancers. However, up to date, no study has inspected the impact of rs10680577 polymorphism on prostate cancer (PCa) risk. This case-control study was achieved on 170 pathologically confirmed PCa patients and 196 cancer free men to inspect whether rs10680577 variant is related to the risk and clinicopathological features of patients with PCa. Genotyping was performed by mismatched polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The findings did not support an association between the variant with the risk and clinicopathological characteristics of PCa patients. When we pooled our results with six preceding studies, the findings suggested that rs10680577 variant significantly augmented the risk of overall cancer in heterozygous (OR=1. hypoxia-inducible factor 1 (HIF-1), a key transcriptional activator is induced by hypoxia (Semenza, 1999). The HIF-1 plays a critical role in the development of solid tumors and in coordinating the cellular response to hypoxia and oxygen homeostasis (Maxwell and Ratcliffe, 2002;Semenza, 2007;Kaelin and Ratcliffe, 2008). The level of HIF-1 is tightly regulated by three PHDs (PHD1, PHD2 and PHD3) (Appelhoff et al., 2004;Willam et al., 2004). In normoxia condition HIF is hydroxylated at specific residues by PHDs which uses oxygen as a substrate. Hydroxylated HIF binds to a protein called Von Hippel Lindau protein (VHL) for its degradation, while in hypoxic situation, stabilization and nuclear translocation occur, leading to oncogenes activation (Appelhoff et al., 2004;Stolze et al., 2006;Pezzuto and Carico, 2018).
Several studies investigated the correlation between EGLN2 4-bp ins/del polymorphism (rs10680577) and susceptibility to various cancer comprising breast cancer (Hashemi et al., 2018b), colorectal cancer (Li et al., 2017), gastric cancer (Wang et al., 2014), hepatocellular carcinoma (HCC) (Zhu et al., 2012), and lung cancer (Che et al., 2014;Zhu et al., 2018), As far as we know, there is no data concerning the impact of EGLN2 4-bp ins/del polymorphism on PCa susceptibility. Consequently, the current study aimed to assess the impact of this variant on PCa development.

Materials and Methods
This case-control study conducted on 170 histologically confirmed PCa patients and 196 cancer free men. The study design and enrollment procedure have been explained previously (Hashemi et al., 2017a;Hashemi et al., 2017b;Sattarifard et al., 2018). The study was approved by the Zahedan University of Medical Sciences ethics committee and all participants were asked to provide their written informed consent. Whole blood samples were collected in EDTA tube, and genomic DNA was purified by salting out method.

Genotyping
Genotyping of EGLN2 4-bp ins/del (rs10680577) polymorphism was done by mismatch polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) as described previously (Hashemi et al., 2018b). The forward and reverse primers were 5`-CCGTTATAAAAGATACTTATGTAAATCAC-3` and 5`-TTGGAATCAAGTGGCGTCG-3`, respectively. PCR was achieved using Prime Taq Premix (Genet Bio, Korea) and the PCR products were digested by AleI restriction enzyme. The del allele digested and created 224 and 31 bp fragments, whereas the ins allele remained undigested (259 bp).

Statistical analysis
All analyses were conducted with SPSS 22 statistical package. The χ 2 and independent sample t-test were used for categorical and continuous data, respectively. Odds ratios (ORs) and 95% confidence intervals (95% CIs) was estimated by logistic regression analysis. P value < 0.05 was considered statistically significant.

Pooled analysis
Pooling of our outcomes with six previous published studies was done using STATA 14.1 software. Electronic databases were searched for all articles describing the relationship between EGLN2 4-bp ins/del polymorphism and cancer susceptibility. The characteristic of study included into pooled analysis is shown in Table 3. The relationship between EGLN2 polymorphism and cancer risk was assessed by pooled ORs and their 95% CIs. The significance of the pooled OR was assessed by the Z-test, and P<0.05 was considered to be statistically significant. Heterogeneity between studies was determined by I2 test and Q test. The I2≥50% or PQ< 0.1 showed the presence of heterogeneity. If heterogeneity exists the random effect model was applied. We determined publication bias using Begg's funnel plot and Egger's test. Sensitivity analyses were conducted in order to assess the data stability.

Results
The study group consisted of 170 histologically confirmed PCa (mean age: 61.2±6.6 years) and 196 cancer free men (mean age: 64.5±8.9 years). Statistically significant difference was observed between cases     Table 4. The Pooled ORs and 95%CIs for the Association between EGLN2 4-bp ins/del Polymorphism and Cancer Susceptibility. specific antigen (PSA) level, Gleason score, perineural invasion, and surgical margin were determined ( Table 2). The results indicated no significant relationship between the variant and clinicopathological features.

Main pooled analysis results
The pooled results with six previous published studies support an association between 4-bp ins/del polymorphism of EGLN2 and cancer susceptibility.  (Table 4 and Figure 1).
Heterogeneity between the studies comprised in the pooled analysis is indicated in Table 2. The findings suggested no heterogeneity in heterozygous and dominant genetic models.
Begg's funnel plot and Egger's test noticed no publication bias in all genetic models except in dominant (Table 4).
We executed sensitivity analysis to evaluate the influence of each study on the overall estimate. The pooled ORs were not substantially changed except in homozygous model, indicating that the present pooled analysis is stable and reliable.

Discussion
Prolyl hydroxylases 1 (PHD1) encoded by EGLN2 gene is involved in the catalyze degradation of HIF-1 Figure 1. The Forest Plot for the Relationship between EGLN2 4-bp ins/del Polymorphism and Cancer Susceptibility for ins/del vs ins/ins (A), del/del vs ins/ins (B), ins/del+del/del vs ins/ins (C), dels/del vs ins/del+del/del (D), and del vs ins (E).
by prolyl hydroxylation of specific residues. Several studies examined the role of EGLN2 4-bp ins/del polymorphism and the risk of some cancers (Zhu et al., 2012;Che et al., 2014;Wang et al., 2014;Li et al., 2017;Hashemi et al., 2018b;Zhu et al., 2018). In the current study, for the first time, we inspected the correlation between EGLN2 4-bp ins/del polymorphism with the risk and clinicopathological characteristic of PCa. Our findings revealed no association between this variant and susceptibility as well as clinicopathological features of PCa patients. Furthermore, pooled analysis of our outcomes with six previous published studies indicated a significant association between the variant and risk of overall cancer in heterozygous, homozygous, codominant, and allele genetic models.
Long non-coding RNAs (lncRNAs), a class of non-coding transcripts longer than 200 nucleotides, are involved in epigenetic, transcriptional and post-transcriptional regulation of gene expression (Ponting et al., 2009). Growing evidence revealed that dysregulation expression of lncRNA contributes to the development and progression of various cancer for their function as proto-oncogene or anti-oncogene (Pibouin et al., 2002;Calin et al., 2007;Lin et al., 2007;He et al., 2016;Tian et al., 2016;Pei et al., 2017).
RERT-lncRNA, with 2,849 base pairs in length, is located within the proximal promoter of EGLN2, and a 4-bp ins/del polymorphism (rs10680577) is within PERT-lncRNA (Zhu et al., 2012). As rs10680577 variant is positioned within the RERT-lncRNA, it is reasonable that this variant may influence the expression level of RERT-lncRNA by affecting its folding structures. Recently, Zhu et al., (2018) reported that 4-bp ins/del polymorphism (rs10680577) affect the expression of EGLN2 and PERT-lncRNA. They found that the ins/ del+del/del genotype carriers had increased expressions level of RERT-lncRNA as well as EGLN2.
In conclusion, our findings proposed that EGLN2 4-bp ins/del polymorphism was not correlated with susceptibility and clinicopathological features of PCa in an Iranian population. Pooled analysis of our findings with previously published studies designated that 4-bp ins/del variant significantly augmented the risk of overall cancer.